Neurodegenerative diseases are characterized by the dysfunction and death of neurons, leading to the loss of neurologic functions mediated by the brain, spinal cord and the peripheral nervous system. These disorders have a major impact on society. For example, approximately 4 to 5 million Americans are afflicted with the chronic neurodegenerative disease known as Alzheimer's disease. Other examples of chronic neurodegenerative diseases include diabetic peripheral neuropathy, multiple sclerosis, amyotrophic lateral sclerosis, Huntington's disease and Parkinson's disease. Normal brain aging is also associated with loss of normal neuronal function and may entail the depletion of certain neurons.
Though the mechanisms responsible for the dysfunction and death of neurons in neurodegenerative disorders are not well understood, a common theme is that loss of neurons results in both the loss of normal functions and the onset of adverse behavioral symptoms. Therapeutic agents that have been developed to retard loss of neuronal activity and survival have been largely ineffective. Some have toxic side effects that limit their usefulness. Other promising therapies, such as neurotrophic factors, are prevented from reaching their target site because of their inability to cross the blood-brain barrier.
Stroke is the third ranking cause of death in the United States, and accounts for half of neurology inpatients. Depending on the area of the brain that is damaged, a stroke can cause coma, paralysis, speech problems and dementia. The five major causes of cerebral infarction are vascular thrombosis, cerebral embolism, hypotension, hypertensive hemorrhage, and anoxia/hypoxia.
The brain requires glucose and oxygen to maintain neuronal metabolism and function. Hypoxia refers to inadequate delivery of oxygen to the brain, and ischemia results from insufficient cerebral blood flow. The consequences of cerebral ischemia depend on the degree and duration of reduced cerebral blood flow. Neurons can tolerate ischemia for 30-60 minutes, but perfusion must be reestablished before 3-6 hours of ischemia have elapsed. Neuronal damage can be less severe and reversible if flow is restored within a few hours, providing a window of opportunity for intervention.
If flow is not reestablished to the ischemic area, a series of metabolic processes ensue. The neurons become depleted of ATP and switch over to anaerobic glycolysis (Yamane et al. (2000) J Neurosci Methods 103(2):163-71). Lactate accumulates and the intracellular pH decreases. Without an adequate supply of ATP, membrane ion pumps fail. There is an influx of sodium, water, and calcium into the cell. The excess calcium is detrimental to cell function and contributes to membrane lysis. Cessation of mitochondrial function signals neuronal death (Reichert et al. (2001) J Neurosci. 21(17):6608-16). The astrocytes and oligodendroglia are slightly more resistant to ischemia, but their demise follows shortly if blood flow is not restored (Sochocka et al. (1994) Brain Res 638(1-2):21-8).
Evidence is also emerging in support of the possibility that acute inflammatory reactions to brain ischemia are causally related to brain damage. The inflammatory condition consists of cells (neutrophils at the onset and later monocytes) and mediators (cytokines, chemokines, others). Upregulation of proinflammatory cytokines, chemokines and endothelial-leukocyte adhesion molecules in the brain follow soon after an ischemic insult and at a time when the cellular component is evolving. The significance of the inflammatory response to brain ischemia is not fully understood (Feuerstein et al. (1997) Ann NY Acad Sci 825:179-93).
Studies in in vivo animal models of stroke, as well as in in vitro paradigms of ischemia-induced neuronal death, have shown that damage and dysfunction of neurons following ischemia is dependent on protein-synthesis (Jin et al. (2001) Ann Neurol. 50(1):93-103; Koistinaho et al. (1997) Neuroreport 8(2):i-viii). Thus, general proteinsynthesis inhibitors such as cycloheximide, and gene transcription blockers prevent ischemia-induced neuronal death (Snider et al. (2001) Brain Res 917(2):147-57). Therefore, the pathophysiology of ischemic stroke involves regulation of gene expression that ultimately result in neuronal death.
The integrated mechanisms of ischemic brain damage and the effect of drug interventions, are readily studied in rodent in vivo models of cerebral ischemia which for these purposes are more suitable than in vitro models. The intact brain preserves the blood-brain barrier and its interactions, and the complex neuronal networks and interactions among neurons and non-neuronal cells. On the other hand, the complexity does not permit detailed studies of particular molecular mechanisms and isolated cellular events. These limitations are overcome in the in vitro models of brain ischemia, were the contribution of blood components are eliminated and tissue temperature, extracellular environment, including ion and nutrient availability, can be standardized. Most in vitro models of ischemia have used a combination of oxygen and glucose deprivation (OGD) to imitate ischemic conditions in vivo (Sick and Somjen (1998) Cerbrovascular disease (Ginsberg M and Bogousslavsky J, eds.), Maiden: Blackwell Science, pp. 137-156). In these studies, the ionic content of the incubation medium, such as the artificial cerebrospinal fluid (aCSF), was similar to that found in normal brains.
However, it is also evident that the in vitro models do not fully reproduce the pathophysiological events that occur in the brain following in vivo ischemia. The hippocampus has been extensively studied following global ischemia in the rat and gerbil, and the damage is characterized by selective neuronal death in the CA1 region appearing 48-72 hours of recovery following 10-15 minutes of ischemia (Kirino (1982) Brain Res 239:57-69; Pulsinelli et al. (1982) Ann Neurol 11:491-498). However, although isolated hippocampal neurons in cultures or hippocampal tissue cultures are readily damaged by OGD, the temporal and special pattern of cell death is not similar to that seen in vivo.
One of the hallmarks of cerebral ischemia is the loss of ion homeostasis across cell membranes due to inhibition of adenosine triphosphate synthesis, which has been studied extensively in animal models of global and focal ischemia (Siesjo (1992) J Neurosurg 77:337-354). The membrane depolarization results in an increase in extracellular potassium, a decrease in extracellular calcium and a decrease in pH (Hansen (1985) Physiol Rev 65:101-148).
In view of the importance of neural disease and ischemia for human mortality and morbidity, the development of suitable models for in vitro screening and development is of great interest.